Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells

Abstract The human tyrosine phosphatase (p54cdc25-c) is activated by phosphorylation at mitosis entry. The phosphorylated p54cdc25-c in turn activates the p34-cyclin B protein kinase and triggers mitosis. Although the active p34-cyclin B protein kinase can itself phosphorylate and activate p54cdc25-c, we have investigated the possibility that other kinases may initially trigger the phosphorylation and activation of p54cdc25-c. We have examined the effects of the calcium/calmodulin-dependent protein kinase (CaM kinase II) on p54cdc25-c. Our in vitro experiments show that CaM kinase II can phosphorylate p54cdc25-c and increase its phosphatase activity by 2.5–3-fold. Treatment of a synchronous population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM kinase II) or the microinjection of AC3-I (a specific peptide inhibitor of CaM kinase II) results in a cell cycle block in G2phase. In the KN-93-arrested cells, p54cdc25-c is not phosphorylated, p34cdc2 remains tyrosine phosphorylated, and there is no increase in histone H1 kinase activity. Our data suggest that a calcium-calmodulin-dependent step may be involved in the initial activation of p54cdc25-c.